Lnc-C2orf63-4-1 Confers VSMC Homeostasis and Prevents Aortic Dissection Formation via STAT3 Interaction

Figures

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  FIGURE 1. Differentially expressed lncRNAs between TAD patients and healthy controls. (A) The hierarchically clustered heatmap illustrating differentially expressed lncRNAs between TAD patients and healthy controls. Up-regulated lncRNAs are denoted in red and down-regulated in green (n = 3 per/group). (B) The volcano plot illustrates the differentially expressed lncRNAs between the TAD patients and healthy controls, with red dots representing up-regulated genes and blue dots representing down-regulated genes (log2fold change ≥2; q ≤ 0.05) (n = 3 per/group). (C–I) qRT-PCR was used to compare the relative expression levels of seven aberrantly expressed lncRNAs (NONHSAT107804.2, ENST00000620266, NONHSAT120405.2, NONHSAT181876.1, NONHSAT070752.2, NONHSAT151355.1, and NONHSAT198707.1) (n = 15 in the control group, n = 24 in the TAD group). The β-actin was selected as a housekeeping gene. Con represents normal aortic tissue from healthy donors; TAD represents aortic dissection tissue from TAD patients. Data (C–I) were expressed as mean ± SEM and compared by Student’s t-test. *p < 0.05.

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  FIGURE 2. The reduced expression of lnc-C2orf63-4-1 is associated with the aortic arch width in clinical TAD patients. (A) RNA-FISH for lnc-C2orf63-4-1 in isolated aorta tissues in TAD patients and healthy controls, clearly suggesting that lnc-C2orf63-4-1 was down-regulated in TAD (n = 3/per group). (B) Schematic annotation of lnc-C2orf63-4-1 genomic locus on chromosome 2q54.972.558-54.972.776 in humans. The length is 218nt. (C) The correlations between the lnc-C2orf63-4-1 expression and ascending aorta diameter of TAD were shown by Pearson’s correlation coefficient (n = 15 in the control group, n = 24 in the TAD group). (D) The correlations between the lnc-C2orf63-4-1 expression and serum D-dimer levels of TAD were shown by Pearson’s correlation coefficient (n = 15 in the control group, n = 24 in the TAD group).

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  FIGURE 3. Overexpression of lnc-C2orf63-4-1 attenuates the AngII-induced apoptosis, fibrosis, and MMP-2/9 activity in VSMCs. (A–C) Lnc-C2orf63-4-1 reduced the expression of Bax and increased the expression of Bcl-2 in mouse VSMCs following AngII treatment (n = 6/per group). (D,E) Apoptosis analysis examined by flow cytometry using Annexin V/PI assay (n = 4/per group). (F–J) Representative WBs and quantification results of MMP-2, MMP-9, type I collagen and type III collagen in the mouse VSMCs treated with or without lnc-C2orf63-4-1 after AngII treatment (n = 6/per group). GAPDH was used for normalization. Data were all compared by analysis-of-variance (ANOVA) with Bonferroni’s post-test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. blank group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. AngII group, & p < 0.05, &&& p < 0.001 vs. AngII + OE-lnc-C2orf63-4-1 group. Data were all expressed as mean ± SEM.

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  FIGURE 4. In vivo targeting of lnc-C2orf63-4-1 reduces AngII-induced AAD in ApoE−/− mice. (A) The expression of lnc-C2orf63-4-1 in aortic tissues in all groups was detected by qRT-PCR (n = 10/per group). U6 was used for normalization. (B) Representative photographs of AAD in male ApoE−/− mice. AngII (1,000 ng/kg/per min) was chronically infused for 28 days, and saline was used as the control. (C) Incidence of AAD in all groups (n = 10/per group). (D,E) Representative pictures from ultrasonography (D) and quantification of the maximal aortic arch (E) of all groups at the end of 28 days saline or AngII infusion (n = 4–10/per group). Data of (A) and (E) were compared by analysis-of-variance (ANOVA) with Bonferroni’s post-test. Incidence of aortic dissection (C) was presented as numbers and compared by using Fisher’s exact test. *p < 0.05, **p < 0.01 vs. control group, ## p < 0.01, ### p < 0.001 vs. AAD group, && p < 0.01, &&& p < 0.001 vs. AAD + OE group. Data were all expressed as mean ± SEM. Con represents saline treatment ApoE−/− mice group; AAD represents aortic dissection and aneurysm model group by ApoE−/− mice treatment with AngII; OE, overexpression of lnc-C2orf63-4-1 by the…

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  FIGURE 5. Overexpression of lnc-C2orf63-4-1 prevents AngII-induced aortic elastin fiber degradation and collagen deposition in aortas of ApoE−/− mice. (A) Representative hematoxylin and eosin staining of the aortas in all groups (n = 4–6/per group). (B) Representative EVG staining of aortic elastin in all groups (n = 4–6/per group). (C) Quantification of aortic elastin degradation in (B). (D,E) Representative Masson’s trichrome staining images and the fibrotic area percent of aorta tissues in all groups are shown (n = 4/per group). Scale bars = 50 μm. Data of (C) and (E) were compared by analysis-of-variance (ANOVA) with Bonferroni’s post-test. **p < 0.01, ***p < 0.001 vs. control group, ## p < 0.01 vs. AAD group, & p < 0.05, && p < 0.01 vs. AAD + OE group. Data were all expressed as mean ± SEM.

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  FIGURE 6. Overexpression of lnc-C2orf63-4-1 inhibits Ang II-induced vascular apoptosis, fibrosis, and MMP-2/9 activity in ApoE−/− mice. (A) Immunofluorescent staining of α-SMA (red) and caspase-3 (green) of the aorta in all groups. (n = 4/per group) (B) Immunofluorescent staining of α-SMA (red) and caspase-9 (green) of the aorta in all groups. (n = 4/per group) (C–F) Representative Western blotting images and quantification of Bcl-2, Bax, MMP-2, MMP-9, type I collagen, and type III collagen in aorta tissue of all groups (n = 6/per group). GAPDH was used for internal normalization. Data of (D–F) were compared by analysis-of-variance (ANOVA) with Bonferroni’s post-test. **p < 0.01, ***p < 0.001 vs. control group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. AAD group, & p < 0.05, && p < 0.01 vs. AAD + OE group. Data were all expressed as mean ± SEM.

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  FIGURE 7. Lnc-C2orf63-4-1 directly interacts with STAT3. (A) Predicted panel of transcription factors potentially interacting with lnc-C2orf63-4-1, exploiting binding free energy below −30 kcal/mol. (B) qRT-PCR measurement of the panel of transcription factors upon AngII with or without lnc-C2orf63-4-1 (AngII + OE). (C) The binding sites of the STAT3 transcription factor with lnc-C2orf63-4-1 were predicted by LncTar (http://www.cuilab.cn/lnctar). (D) Detection of STAT3 mRNA using qRT-PCR in the aorta samples of healthy donors and clinical TAD patients (n = 6–10/per group). (E) Representative Western blotting images and quantification of STAT3 in aorta samples of healthy donors and clinical TAD patients (n = 6–10/per group). GAPDH was used for internal normalization. (F) The expression level of STAT3 in aorta tissue of all groups was evaluated by qRT-PCR (n = 4–6/per group). β-actin was used for normalization. (G) Representative Western blotting images and quantification of STAT3 in aorta tissue of all groups (n = 4–6/per group). GAPDH was used for internal normalization. (H) Dual-luciferase reporter assay of WT and Mut STAT3 3′UTR. (n = 4/per group). *p < 0.05 vs. Vector group. (I)…

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  FIGURE 8. Lnc-C2orf63-4-1 restores apoptosis, fibrosis, and MMP-2/9 activity via destabilizing STAT3 mRNA in VSMCs. (A,B) qRT-PCR analysis results showed that lnc-C2orf63-4-1 silencing by lentivirus vector carrying a lnc-C2orf63-4-1-shRNA fragment promoted the expression of apoptosis-related markers via induction of the STAT3 (n = 6/per group). (C,D) qRT-PCR analysis results showed that lnc-C2orf63-4-1 silencing by lentivirus vector carrying a lnc-C2orf63-4-1-shRNA fragment promoted the expression of MMP-2 and MMP-9 via induction of the STAT3 (n = 6/per group). (E,F) qRT-PCR analysis results showed that lnc-C2orf63-4-1 silencing by lentivirus vector carrying a lnc-C2orf63-4-1-shRNA fragment promoted the expression of fibrosis-related markers via induction of the STAT3 (n = 6/per group). (G) Half-life (t1/2) of STAT3 mRNA in lnc-C2orf63-4-1 treatment by lentivirus vector carrying the lnc-C2orf63-4-1 sequence in mouse VSMCs. VSMCs were pre-incubated with actinomycin D (ActD, 5 μg/ml), then treated with lnc-C2orf63-4-1 for different time intervals (n = 6/per group). The data were presented as means ± SEM from four independent experiments in duplicate. *p < 0.05, **p < 0.01, ActD +…